Conductive polymers have been previously used in chemo- and biosensors (see Lange et al, Anal. Chim. Acta, 614, 2008). All conductive polymers exhibit and intrinsic affinity for redox-active compounds and many exhibit an affinity for acidic or basic gases and solvent vapours. In order to achieve a specific interaction with the analyte, polymers may be modified with receptors. Receptors that have been immobilised to polymers exhibit an important advantage compared to other receptors. The conductive polymer wire enables a collective response, which leads to a significant increase in signal. Swager et al (Acc. Chem. Res., 31, 1998) demonstrate that a conjugated polymer receptor for methylviologen exhibits a 65 times larger signal amplification in comparison to a monomer receptor.
The application of conductive polymers for chemical- and biosensors can be implemented using various methods of detection. A detailed discussion of these aspects can be found in Lange et al, Anal. Chim. Acta, 614, 2008.
The most commonly used technology for signal transduction for chemiresistor sensors based on conducting polymers is the measurement of conductivity. Examples of such applications can be found in the following patents: US 2004040841, JP 2003302363, US 2007114138, US 2007029195, US 2003148534, US 2004202856, U.S. Pat. No. 6,493,638, DE 102004047466, RU 2174677, U.S. Pat. No. 5,869,007, U.S. Pat. No. 5,337,018 and U.S. Pat. No. 5,882,497. This technology exhibits various advantages: 1) small disruptions along the polymer chain can change the conductivity of the whole chain. Therefore, an increased sensitivity can be obtained in comparison to electrochemical or optical methods of measurement. 2) The measurement set-up is relatively simple, although enables a precise and highly sensitive measurement. 3) Measurements of conductivity can be implemented using nano wires of conductive polymers. 4) Single chemiresistors can easily be combined into arrays. 5) With the help of RFID technology, such measurements can be carried out without wire connection. There are multiple possible configurations that can be applied to in order to measure the conductometric responses of such sensors. Some of them are summarised in FIG. 1.
In 2-point technology (FIG. 1a) the conductive material is applied to two electrodes that are separated by a narrow gap. The conductivity is then measured by the application of a constant current or voltage (dc or ac) between the electrodes and the resulting voltage or current is then measured. The more rarely used 4-point measurement technology measures the conductivity of the conductive layer without influence of the voltage drop between the conductive layer and the metal contacts (FIG. 1d). This technology has recently been modified so that the 2- and 4-point technologies have been combined (FIG. 1e).
A further possibility is the use of organic field transistors as sensors. Here the current is controlled between the source and the drain electrode by the gate voltage. Based on the similarity of the measurement configuration, Wrighton et al (J. Am. Chem. Soc., 1990) introduced the term electrochemical transistor for an apparatus based on a conductive polymer, in which the redox-state of the polymer is controlled by the voltage between the working electrode (source and drain) and a reference electrode (gate) (FIG. 1c, f). The measurements of electrical current between two or more electrodes on a substrate is the most practical method, however the low conductivity of the chemically sensitive films under conditions typical for bioanalytical measurements (neutral ph, low oxidation potential) can complicate these methods. In such cases measurement of the resistance between an electrode, which is modified with a chemically sensitive film, and another electrode in a solution, is often used (FIG. 1b). These measurements can be carried out using the commonly known 2- or 3-electrode configurations. One advantage of such measurements is that the ratio of electrode surface to film thickness is approximately 100 times greater, which leads to an approximately 100 times lower absolute resistance in comparison to the resistance of lateral measurements.
In most chemiresistors, changes in conductivity are measured using the 2-point technology. A low voltage direct current (approximately 5-100 mV) or a constant current is applied between the two electrodes, which are separated by a narrow gap, and the resulting current/voltage drop is measured. Such micro electrodes are most often produced with help of photolithography. Approximately 50-200 nm thick gold or platinum films are deposited onto oxidised silicon glass surfaces, using a very thin adhesive layer. The gap between the electrodes is normally between 1.5 μm and 100 μm, although smaller gaps are preferred due to the smaller amount of chemically sensitive material that must be used in order to cover the gap, which causes increased sensitivity. When the material is separated over the gap, larger gaps commonly lead to thicker polymer films and therefore to a loss of sensitivity. The pre-treatment of the electrode with for example hydrophobic silanes can enhance the growth of the polymer over the gap, whereby the pre-treatment of gold electrodes with thiol monomers enhances contact between the electrode and the polymer film and therefore reduces the influence of the contact resistance.
The application of a constant voltage or a constant current in order to measure changes in conductivity of sensors based on chemiresistors leads to various problems. Irreversible or slowly reversible changes in the film can occur, which can be avoided by the application of alternating current voltage or oppositely polarised direct current pulses. Additionally, a high voltage through a chemically sensitive film leads to heating of the film, which leads to the conductivity becoming sensitive to parameters such as air- or liquid flow. Another reason for maintaining the voltage as low as possible is the non-linearity of the current-voltage curve. However in contrast the signal to noise ratio increases with increasing voltage. Therefore a compromise must be found between a voltage that is high enough to produce an appropriate signal to noise ratio and the effect of heating.
Alternating current measurements have one advantage over direct current measurements. It has been reported that the noise during conductivity measurements exhibit a flicker noise behaviour and a decrease of 1/f (whereby f is the frequency) during increasing frequency. This noise behaviour was explained by a contribution of the contact resistance between single polymer grains on the total resistance of the polymer film. This intermediate grain resistance has a parallel switched capacity, which bridges the resistance at higher frequencies. Additionally, impedance analyses can be made for alternating current measurements, or simultaneous resistive and capacitive changes can be measured, which increases the sensor selectivity. A sensor that retrieves various frequencies has been reported. Measurements of a few selective frequencies are typically much faster than the measurement of impedance.
The use of multiple electrodes and the switching between electrodes enables an increase in the measurement through flow. Slater et al (Analyst, 118, 1993) used such multiple electrodes in order to create a multi layer gas sensor. Regarding measurement of electrical conductivity between electrodes at different distances, the authors proposed that it is possible to order or allocate the conductive zones of the polymers. Subsequently, the conductivity between electrodes positioned close together is most substantially influenced by a thin polymer layer which is close to the electrode substrate. Thin films were also found to be more sensitive to gases when compared to thick films.
The resistance measured by the 2-point technology includes the polymer resistance and the resistance between the contacts in the polymers. When the contact resistance is high, and in comparison with the polymer resistance shows only changes regarding the analytes, it can limit the sensitivity of the system. Most synthesis technologies for thin chemosensitive films lead to the creation of micrometer or submicrometer thick layers. Therefore the method from Cox and Strack (Solid-State Electron, 10, 1967), which is based on the change in the relationship between a contact area to the thickness of the material, can hardly be used in order to distinguish between the polymer and the contact resistances.
Importantly, this can be achieved through impedance spectroscopy, whereby broad frequency spectrums are measured, therefore enabling separation of the resistance of the polymer and the resistance between the polymer and the metal contacts. However, these measurements are relatively slow because a broad frequency band must be covered. Additionally, the results of the impedance spectroscopy are influenced by the selection of the electrical circuit for the data analysis. This problem can however be solved more easily using 4-electrode configuration-based measurement technologies.
In the 4-electrode configuration, the conductivity is measured by the application of constant voltage between two outer electrodes, whereby the difference in potential between two inner electrodes is measured. This difference in potential is measured using a voltmeter with high input resistance and is therefore not influenced by the drop in ohmic potential in response to the contact resistance of the inner electrodes. Four parallel metallic strips are commonly used as contacts for the 4-electrode configuration. For a more effective usage of the sensor surface when using the 4-point measurement, wound electrodes have been developed. Such electrodes have been used as single sensors and as arrays, comprising of 96 such electrodes, and enable a combinatorial investigation of material.
If the contact resistance is so high that it limits the dynamic range of the sensor signal, the application of a 4-electrode configuration of a higher sensitivity is required. On the other hand, 4-electrode measurements alone provide no information on the resistance of the contacts between film and metal. Many processes lead to the detachment or displacement of the film from the contacts or to the formation of poor conducting contacts. For example the common process of chemical immobilization of proteins has been reported to lead to a detachment of the polymer. On the other hand, the contact resistance can contain additional analytical information which can be useful for analysis.
The simultaneous 2- and 4-point resistance measurement (S24) provides a possibility to measure the 4- in addition to the 2-point resistance, whereby the subtraction of both values leads to the determination of the contact resistance. The relationship of the resistances, which are measured through the 2- and 4-point technologies, provides valuable information regarding the quality of the contact between middle and conductive film. The calculation of quantitative values from such measurements is based on the following idea. The 2-point technology provides the R2 value, which is the sum of the film resistance in the two contact resistances, whereby the 4-point technology measures the film resistance R4 between the middle points of the inner electrodes.
The contact resistance can therefore be calculated by Rc=R2−α·R4, whereby α is the geometric factor. When the electrode geometry is the same for all examined polymers, the constant α can be used in order to calculate the contact resistance from the values R2 and R4. It has been demonstrated that small variations in α do not lead to qualitative changes in the behaviour of the contact resistance. This approach has been used to study the dependence of contact resistance between polypyrol and gold electrodes under various electrode potentials. These results have been compared with the results from impedance-spectroscopy. A strong agreement has been observed for a change of 4 orders of magnitude.
A number of other methods for quantitative estimation of the contact resistance have been described. This value can be obtained, for example, by switching between 4- and 3-electrode configurations. Measurements of the electric potential between the source and sense electrodes and the straight-line extrapolation of the drop in voltage allow one to calculate the voltage drop at the source and drain contacts. An examination of the resistance' dependence on the distance between two electrodes and the calculation of the contact resistance through the following extrapolation of this dependence has also been reported.
Monitoring of contact resistance or the ratio of the two- and four-point resistances allows us to make internal control of the sensor integrity: a desorption of sensor molecules from the electrode can be detected and thus distinguished from increase of the resistance of chemosensitive material.
One disadvantage of these methods of measurement is that they do not allow controt of the redox-state of the polymer or of any other chemosensitive material. This can however be carried out electrically through the fixing of the potential of the chemosensitive material in relation to a reference electrode. Such measurements are often designated as in-situ resistance measurements and the measurement configurations are commonly named electro-chemical transistors. This naming has been accepted in the literature and is therefore used herein. However such terms should not be confused with semi-conductors or organic transistors, which function in a different manner.
The first application of electrochemical transistors was described by White et al. in 1984. They use a symmetrical configuration, comprising three gold electrodes of three micrometers that were separated by a 1.4 micrometer-wide gap. The central electrode which was used to control the redox-state of the polymer was bound as a working-electrode with a potentiostat. Auxiliary- and reference electrodes were immersed in solution. Due to the similarity to field effect transistors the central gold microelectrode was designated “gate” and the two other electrodes designated “source” and “drain”. This configuration has been further simplified by leaving out central electrode, whereby the control of the redox-state of the film is carried out by through the source or drain electrode. This allows an even smaller gap between the source and the drain electrode and therefore a faster reaction time. However with highly specific polymer resistance this asymmetric configuration can lead to variation or deviation in the condition of the polymer close to the second electrode and therefore to heterogeneous polymer properties between the source and drain electrodes.
A further simplification of the configuration of the electro-chemical transistor can be carried out by the replacement of the potentiostat with a reference electrode and serially connected potential source. However in this case the ohmic voltage drop at a low polymer resistance can lead to deviation from the applied potential. A difference in potential between the source and drain electrodes can be achieved by the application of a bipotentiostat. In this case the potentials of the source and drain electrodes are simultaneously controlled and a constant small difference in potential is maintained. The current through the drain and source electrodes comprise of faraday units, which are induced by the redox-reaction of the polymer and an ohmic component produced through the difference in electric potential between the electrodes.
An alternative technology relates to the combination of common 2- or 4-electrode configurations for the measurement of resistance with a reference electrode or using a potentiostat. When a constant voltage between the source and the drain electrodes is applied the measured current contains not only the current between the 2 electrodes but also the current between the film and the auxiliary or the reference electrode in the electrolyte. In order to solve this problem, voltage pulses with opposite polarity and low frequency (<1 Hz) or a triangular voltage pulse can be used. In such cases various methods are used in order to calculate the polymer resistance. Wrighton and colleagues calculate the increase in the I-V-curve at zero voltage (Paul et al. J. Phys. Chem. 89, 1441, 1985). When pulses instead of triangular wave forms are used, the voltage is usually measured at the end of each pulse, when transistent effects are minimal. In Kruszka et al (Rev. Sci. Instrum. 62, 695, 1991) the voltage from three consecutive measurements was calculated, whereby the current of the negative pulse was subtracted from the average current of the two positive pulses. The averaging of the positive and negative pulses provides almost the same results. The removal of current that traverses the electro-chemical cell is especially important when the current between the source and drain is very small.
In order to apply such electro-chemical transistors in sensor applications the factors which influence the in-situ-resistance should be considered. Such in-situ-measurements of resistance are commonly used to study thin conductive polymer films. This technology is often coupled to other technologies such as cyclic voltammetry, ESR or quartz crystal micro balance. However in most studies the 2-point resistance measurement is applied. The application of simultaneous 2- and 4-point measurements demonstrates that the data which is obtained through the 2-electrode configuration may contain significant mistakes regarding the contact resistance, especially regarding high polymer conductivity.
A modification of chemosensitive materials with enzymes, which directly interact with the material or release substances during the enzymatic cycle that influences the material resistance, represents a simple possibility for producing conductometric enzymatic biosensors. External references and auxiliary electrodes can be used in the simplest constructions (FIG. 1c), however these electrodes can not be directly applied to the microchip surface.
Solid state electro-chemical transistors were first reported by Chao (J. Am. Chem. Soc. 109, 6627, 1987). Such transistors exhibit a solid electrolyte which is precipitated over an array, which comprises of 8 microelectrodes. Two of these electrodes were used as counter electrodes, whereby the other 6 were bound to a conductive polymer film. A drop of silver adhesive was used as the reference electrode. The chip was covered with polyvinyl alcohol and used as a humidity sensor.
The influence of faradaic and non-faradaic processes on the gate electrode in regards to the performance of the electrode chemical transistors has been examined. The charge, which is necessary for the oxidation (doping) of the conductive polymer can be compensated for by the discharge of the double-layer of the gate electrode (non-faradaic process) or by a reduction process on the gate electrode (faradaic process). However the relatively small capacity of the ionic double-layer can limit the charge of the polymer oxidation. Faradaic processes have a much higher pseudo-capacity, which leads to a higher sensitivity of the transistor voltage in response to changes in the gate potential. In order to enable faradaic processes of the gate electrode, the gate electrode can be covered with a redox-active material. A large surface relationship between the gate and the work-electrode reduces the switching time.
The switching time and the charge of the transistor depend strongly on the amount of the polymer, which is necessary to bridge the gap between the source and the train electrode. A switching point of 0.1 milliseconds has been reported for PANI when using a gap of 50-100 nm. This construction was switched under the electrical charge of 1 nC. For a similar construction with 1.5 micrometer wide gap, approximately 100 times higher charge was required. Constructions with very small gaps are also used in sensors. The modification of polyanaline with glucose oxidise has been produced for in vivo applications as a biosensor for glucose.
Electrochemical transistors are produced from conductive polymers on flexible substrates. In this case PEDOT/PSS is used as a contact, channel and gate material. Fast switching times at low air humidity could be achieved, whereby a very hygroscopic solid electrolyte was used, which comprises of PSS, ethylene glycol, sorbitol and LiClO4. There were various measurement configurations for such transistors proposed. The most simple is a 3 electrode electrochemical transistor. A solid electrolyte covers the channel between the source and outlet electrodes and the gate electrode. In modification of this configuration can be achieved when references are attached to a second gate electrode, which is in contact with the channel. This is a configuration similar to others whereby a central electrode is positioned as a gate electrode between the source and drain electrodes. This transistor was tested as a humidity sensor. The changes in conductivity of the channel at the gate voltage of 1.2 V depended strongly on the humidity-dependent conductivity of the solid electrolyte. In another configuration both gate electrodes are not in contact with the channel.
There are various problems with affinity sensors which are based on conductive polymers or other chemiresistive materials. Firstly, desorption of an analyte can be very slow. In order to accelerate this process the sensor can be heated (see e.g. DE 102004047466). However this can lead to damages of the chemosensitive material and it is time intensive to bring the sensor to the correct working temperature. The electric control of the affinity allows a different solution to this problem. Many chemosensitive materials (for example polyaniline, polypyrrol and their derivatives) demonstrate similar responses to changes in their protonation states and oxidation states which complicates the application of these materials as pH sensors. The electric control of the chemosensitive materials enables controlling the oxidation state.
Distinguishing between contact and film resistance of the polymer or any other chemosensitive material requires the application of the 4-point configuration for the resistance measurement, whereby additionally one reference or two reference electrodes can be applied in order for the redox state of the sensor material to be controlled. This approach has not been described in any prior art in regards to chemo resistors based on organic or inorganic conductive polymers.
A further aspect of the invention relates to the application of conductive polymers as optical signal transformers (e.g. US 2003148534, JP 4190142, JP 60202334, DE 10254841, G01N31/00, G01N21/55, G01N21/77, G01N31/22, G01N31/00, G01N21/55, G01N21/77, G01N21/77, G01N27/12). A typical optical detection method is based on changes in the absorption spectrum in the range between ultra violet and near infrared; however in some cases fluorescent or refractometry based detections methods could be applied (surface plasmon resonance). Also in these cases a regeneration of the highly sensitive sensor, through flushing with either gas or solution without analyte, is very slow and the chemical properties of the chemosensitive material could be modified via spontaneous oxidation or reduction during storage before application.